The broad longterm objective of this research is a complete understanding of store-operated Ca2+ entry, a process that underlies sustained physiological Ca2+ signalling in many types of cells. It has particular importance in the activation of T cells and other immune system cells, and hence is a therapeutic target in autoimmune diseases. Very recent research has uncovered an important role for store-operated Ca2+ influx in the development, progression, and invasiveness of a variety of solid tumors. The key cellular controllers of store-operated Ca2+ influx, STIM1 and STIM2, were identified in 2005, and the Ca2+ channel ORAI1 and its paralogues ORAI2 and ORAI3 were identified in 2006. There have been only limited studies, however, on cellular proteins that regulate the pathway. This specific proposal arises from a genome-wide RNAi screen that identified dozens of previously unrecognized regulators of store-operated Ca2+ entry, among them the filamentous proteins septins 4 and 5. Septin filaments had been known to demarcate certain specialized subregions of the plasma membrane and to serve as scaffolds for signalling proteins, but they had not been connected to Ca2+ signalling. Further experiments led to a conclusion that septins govern the stability of STIM-ORAI channel clusters at ER-plasma membrane junctions. A local rearrangement of septins and the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate plays a critical role. These findings highlight the need to understand the stability of ORAI at signalling clusters and the dynamic changes at ER-plasma membrane junctions during Ca2+ signalling. The project will use conventional confocal microscopy and advanced imaging techniques including TIRF microscopy and single-particle tracking, along with judicious protein engineering, to dissect the protein and lipid rearrangements and local signalling processes at ER-plasma membrane junctions that control and modulate STIM-ORAI signalling. The aims are (1) to determine how septins, along with other proteins and the local lipid microdomain, constrain ORAI movements at ER-plasma membrane junctions and thereby stabilize the STIM- ORAI complex; (2) to examine dynamic changes in the spatial distribution of phosphoinositides in the ER- plasma membrane junction during Ca2+ signalling, and the importance of these changes for STIM-ORAI signalling; and (3) to define the role of the ER-membrane proteins TRIM59 and TMEM110, regulators of Ca2+ influx identified in the RNAi screen, that, like STIM1, move to ER-plasma membrane junctions during Ca2+ signalling. This research will provide important insights into the regulation of physiological Ca2+ signals, and may uncover new targets for therapeutic intervention in autoimmune disease and cancer.